The present invention relates to the inhibition of vascular smooth muscle cell proliferation using a polynucleotide encoding a xe2x80x9csuicidexe2x80x9d gene. When expressed intracellularly, and stimulated by a second compound, the product of the suicide gene kills proliferating cells.
The response to arterial injury in vivo is mediated by a complex set of cellular interactions involving endothelial and smooth muscle cells. Following damage to the arterial wall, growth factors and cytokines are released locally and induce cellular proliferation through autocrine and paracrine mechanisms. A common and clinically significant setting for such injury is balloon angioplasty wherein blood vessels narrowed by atherosclerotic deposits are opened using an inflatable balloon. Dilation of the occluded vessel can result in a reactive cellular proliferative response which leads to renarrowing (restenosis) of the arterial lumen. Blood flow is compromised by hyperplasia of the intimal (adjacent to the lumen) layer of the artery and to deposition of extracellular matrix components. Restenosis occurs in approximately 30% of coronary artery angioplasties, thereby presenting a major roadblock to the successful treatment of cardiovascular disease.
A number of approaches for controlling smooth muscle cell proliferation following angioplasty have been attempted, including angiotensin converting enzyme (ACE) inhibitors and antisense RNA directed against cell cycle control proteins (Rakugi et al., (1994) J. Clin. Invest., 93:339-346; Simons et al., (1992) Nature, 359:67-70). Although these pharmacological approaches have been somewhat effective in preventing the neointimal hyperplasia associated with balloon angioplasty in a rat carotid model, the application of these approaches to human disease has been unsuccessful.
Replication-deficient adenoviral vectors have been used in a number of promising approaches to gene therapy. Lemarchand et al. demonstrated transfer of the xcex2-galactosidase and xcex11-antitrypsin genes into the endothelium of normal arteries and veins in sheep using an adenoviral vector (Circulation Res., 5:1132-1138, 1993; Proc. Natl. Acad. Sci. USA, 89:6482-6486, 1992). Lee et al. (Circulation Res., 73:797-807, 1993) demonstrated adenoviral vector-mediated transfer of the xcex2-galactosidase gene into balloon-injured rat carotid arteries. These vectors have also been used to transduce mouse hepatocytes in vivo (Stratford-Perricaudet et al., (1990), Hum. Gene Ther., 1:241-256). In addition, expression of a recombinant xcex2-galactosidase gene has been observed after infusion of an adenoviral vector into rabbit coronary arteries (Barr et al., (1994) Gene Therapy, 1:51-58).
Culver et al. (Science, 256:1550-1552, 1992) injected murine fibroblasts expressing the herpes simplex virus thymidine kinase (HSV-tk) gene into rats with a cerebral glioma. The rats were then given the nucleoside analog ganciclovir (GCV). Once GCV entered the cells expressing the HSV-tk gene, it was phosphorylated by the newly expressed thymidine kinase. Cellular kinases can also phosphorylate GCV, which is incorporated into replicating DNA (Smith et al., (1982) Antimicrob. Agents Chemother., 22:55-61) and causes premature chain termination. As this process inhibited DNA replication, only the actively dividing cells were killed. In this experiment the gliomas regressed completely both microscopically and macroscopically. Other nucleoside analogs capable of being modified by thymidine kinase, such as acyclovir (Elion et al., (1977) Proc. Natl. Acad. Sci. U.S.A., 74:5716-5720), have been used as targets for suicide inhibition of cellular replication.
Moolten et al. (Hum. Gene Ther., 1:125-134, 1990) induced lymphomas with Abelson leukemia virus in transgenic mice carrying the HSV-tk gene. Following treatment of 12 mice with GCV, 11 exhibited complete tumor regression.
Plautz et al. demonstrated in vivo regression of a transplantable murine adenocarcinoma transfected with a HSV-tk gene and treated with GCV. In these same experiments, expression of a HSV-tk-xcex2-galactosidase construct in nondividing rabbit arterial cells was unaffected by GCV treatment, demonstrating the selectivity of this approach in the maintenance of quiescent cells and the elimination of rapidly dividing cells in vivo (Plautz et al., (1990) New Biologist, 3:709-715).
The efficacy of introducing a suicide gene into smooth muscle cells has not been previously addressed. For this reason, there exists a need for safe, effective methods of inhibiting neointimal hyperplasia after mechanical vessel injury. The present invention provides a solution to this need.
One embodiment of the present invention is a method for inhibiting restenosis associated with mechanical treatment of a blood vessel in a mammal comprising:
introducing a polynucleotide encoding a thymidine kinase gene to the blood vessel after mechanical treatment;
expressing the thymidine kinase gene to produce thymidine kinase protein in cells of the blood vessel; and
then administering to said mammal an effective amount of a DNA replication-inhibiting nucleoside analog capable of being phosphorylated by the thymidine kinase protein and preferentially incorporating the phosphorylated analog into the DNA of proliferating cells, whereby the proliferating cells are killed.
Preferably, the mechanical treatment is balloon angioplasty, laser, atherectomy device or stent implantation and the thymidine kinase gene is in a eukaryotic expression vector. More preferably, the expression vector is a viral vector. Most preferably, the viral vector is an adenoviral vector. In another aspect of this preferred embodiment, there is provided a polyoma virus enhancer, adenoviral vector enhancer elements, encapsidation signals and an origin of replication separate from said thymidine kinase gene. In a particularly preferred embodiment, the adenoviral vector is Ad.HSV-tk. In another aspect of the invention, the expression vector is complexed with a nonviral vector. Preferably, this nonviral vector is a liposome or receptor ligand. Advantageously, the suicide compound is either ganciclovir or acyclovir and the modification is phosphorylation. In another aspect of this embodiment, the phosphorylated compound is further phosphorylated by cellular enzymes and is preferentially incorporated into the DNA of rapidly dividing cells.
The present invention also provides a recombinant adenoviral vector Ad.HSV-tk comprising:
a wild type adenovirus wherein the E3 region and about 9 map units have been deleted; and
a HSV-tk expression cassette inserted into the deleted region, the expression cassette comprising the herpes simplex virus thymidine kinase gene operably linked to promoter, enhancer, encapsidation signal and origin of replication elements. Preferably, the wild type adenovirus is type 5 adenovirus and the elements are derived from polyoma virus and adenovirus.
In another aspect of the invention, there is provided a method for inhibiting restenosis associated with mechanical treatment of a blood vessel in a mammal comprising:
introducing a polynucleotide to the blood vessel after the mechanical treatment, the polynucleotide comprising a suicide gene that encodes a suicide protein;
expressing the suicide gene to produce the suicide protein in cells of said blood vessel; and
administering a suicide compound to a mammal, wherein the proliferating cells are killed as a result of modification of the suicide compound by the suicide protein.
In another aspect of this embodiment, the mechanical treatment is balloon angioplasty, laser, atherectomy device or stent implantation. Preferably, the suicide gene is the thymidine kinase gene and the suicide protein is thymidine kinase. Advantageously, the suicide gene is contained within a eukaryotic expression vector, preferably a viral vector. In another aspect of this preferred embodiment, the viral vector is a retroviral vector; most preferably, it is an adenoviral vector. In another aspect of the invention, the eukaryotic expression vector containing the suicide gene may be complexed with nonviral vectors such as liposomes or receptor ligands. In preferred embodiments, the suicide compound is ganciclovir or acyclovir and is phosphorylated by the thymidine kinase. In a particularly preferred embodiment, the phosphorylated ganciclovir is preferentially incorporated into the DNA of rapidly dividing cells.